Publications by authors named "Durvanei A Maria"

33 Publications

Comparison between placental and skeletal muscle ECM: implantation.

Connect Tissue Res 2020 Oct 26:1-14. Epub 2020 Oct 26.

Department of Surgery, Sector of Anatomy, School of Veterinary Medicine and Animal Science, University of São Paulo , São Paulo, Brazil.

Purpose Of The Study: Several tissues have been decellularized and their extracellular matrices used as allogeneic or xenogeneic scaffolds, either in orthotopic or heterotopic implantations, for tissue engineering purposes. Placentas have abundant matrix, extensive microvascular structure, immunomodulatory properties, growth factors and are discarded after birth, representing an interesting source of extracellular matrix. This study aimed at comparing decellularized canine placentas and murine skeletal muscles to regenerate skeletal muscles in a rat model.

Materials And Methods: Muscle pockets were created at the posterior limbs of male Wistar rats, where the muscle- and placenta-derived extracellular matrices were implanted. Macroscopic, histological, and immunohistochemical analyses were performed after 3, 15, and 45 days of surgeries.

Results: On the third day, intense inflammatory reaction, with macrophages (CD163) and proliferative cells (PCNA) being observed in control group and adjacent to the decellularized matrices. The percentage of proliferative cells was higher in placenta than in muscle matrices. Macrophages CD163 were higher in muscles than in placentas, whereas CD163 were higher in placentas than in muscle ECM, at days 3 and 15. Placental matrices were not completely degraded at day 15, as opposed to the muscular ones. After 45 days, both matrices were resorbed and morphologically normal myofibers, with reduction of cell infiltration, were observed.

Conclusions: These results demonstrated that xenogeneic placental ECM, implanted heterotopically (representing a biologically critical and challenging microenvironment), induced local inflammatory reactions similar to the allogeneic muscle ECM, implanted orthotopically. Thus, placenta-derived extracellular matrix must be further explored in regenerative medicine.
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http://dx.doi.org/10.1080/03008207.2020.1834540DOI Listing
October 2020

Laser Photobiomodulation Over Teeth Subjected to Orthodontic Movement.

Photomed Laser Surg 2018 Dec;36(12):647-652

Movement Laboratory of Nove de Julho University, Santana, São Paulo, Brazil.

Orthodontics of the 21st century requires aesthetic, painless, predictable, and quick treatments. This demand for faster results generated orthodontic movement acceleration protocols (OMAPs); among other OMAPs we present low-level laser (LLL) as a candidate. To evaluate levels of interleukin (IL)-1, IL-10, and type 1 collagen in the periodontal ligament of first molars of rats subjected to orthodontic traction with and without LLL irradiation, compared with untreated controls (CO), and to evaluate whether the dose of LLL used in this work is eligible as an OMAP. A total of 35 male Wistar rats were distributed into three groups: group 1 NI (nonirradiated)  = 15, group 2 IR (laser irradiated using 5 J, 177 J/cm, and 100 mW applied in contact to the vestibular mesial, vestibular distal, and palatal faces of gum tissue around molar region for 50 sec each point, for 3 consecutive days, immediately 24 and 48 h after orthodontic device placement.)  = 15, and group 3 CO  = 5; groups 1 and 2 were subjected to orthodontic force and each group was divided into three subgroups that were sacrificed after 3, 5, and 7 days, IL-1/10 and COL-1 levels were analyzed. In the IR group, levels of IL-1/10 and COL-1 showed peak anticipation after LLL irradiation compared with those in the NI and CO groups. These results can also infer that this dose of LLL can be used as an OMAP.
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http://dx.doi.org/10.1089/pho.2018.4532DOI Listing
December 2018

Preclinical evaluation of Amblyomin-X, a Kunitz-type protease inhibitor with antitumor activity.

Toxicol Rep 2019 1;6:51-63. Epub 2018 Dec 1.

Laboratório de Biologia Molecular, Instituto Butantan, Av. Vital Brasil, 1500, São Paulo, 05503-900, SP, Brazil.

Amblyomin-X, a Kunitz-type protease inhibitor, is a recombinant protein that selectively induces apoptosis in tumor cells and promotes tumor reduction in melanoma animal models. Furthermore, Amblyomin-X was able to drastically reduce lung metastasis in a mice orthotopic kidney tumor model. Due to its antitumor activity, Amblyomin-X potential to become a new drug is currently under investigation, therefore the aim of the present study was to perform preclinical assays to evaluate Amblyomin-X toxicity in healthy mice. Exploratory toxicity assays have shown that treatment with 512 mg/kg of Amblyomin-X lead to animal mortality, therefore two groups of treatment were evaluated in the present work: in the acute toxicity assay, animals were injected once with doses ranging from 4 to 256 mg/kg of Amblyomin-X, while in the subacute toxicity assay, animals were injected with 0.25, 0.57 and 1 mg/kg of Amblyomin-X daily, during 28 days. Following this treatment regimens, Amblyomin-X did not cause any mortality; moreover, toxicity signs were discrete, reversible and observed only at the higher doses, thus establishing a safety profile for administration in mice, which can be further used to determine the dose translation of this novel drug candidate for treatment in other species.
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http://dx.doi.org/10.1016/j.toxrep.2018.11.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298944PMC
December 2018

Cell viability assessed in a reproducible model of human osteoblasts derived from human adipose-derived stem cells.

PLoS One 2018 11;13(4):e0194847. Epub 2018 Apr 11.

Department of Internal Medicine, Botucatu Medical School, São Paulo State University - UNESP, Botucatu, São Paulo, Brazil.

Human adipose tissue-derived stem cells (hASCs) have been subjected to extensive investigation because of their self-renewal properties and potential to restore damaged tissues. In the literature, there are several protocols for differentiating hASCs into osteoblasts, but there is no report on the control of cell viability during this process. In this study, we used osteoblasts derived from hASCs of patients undergoing abdominoplasty. The cells were observed at the beginning and end of bone matrix formation, and the expression of proteins involved in this process, including alkaline phosphatase and osteocalcin, was assessed. RANKL, Osterix, Runx2, Collagen3A1, Osteopontin and BSP expression levels were analyzed using real-time PCR, in addition to a quantitative assessment of protein levels of the markers CD45, CD105, STRO-1, and Nanog, using immunofluorescence. Rhodamine (Rho123), cytochrome-c, caspase-3, P-27, cyclin D1, and autophagy cell markers were analyzed by flow cytometry to demonstrate potential cellular activity and the absence of apoptotic and tumor cell processes before and after cell differentiation. The formation of bone matrix, along with calcium nodules, was observed after 16 days of osteoinduction. The gene expression levels of RANKL, Osterix, Runx2, Collagen3A1, Osteopontin, BSP and alkaline phosphatase activity were also elevated after 16 days of osteoinduction, whereas the level of osteocalcin was higher after 21 days of osteoinduction. Our data also showed that the cells had a high mitochondrial membrane potential and a low expression of apoptotic and tumor markers, both before and after differentiation. Cells were viable after the different phases of differentiation. This proposed methodology, using markers to evaluate cell viability, is therefore successful in assessing different phases of stem cell isolation and differentiation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194847PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5895002PMC
July 2018

Cell internalization of 7-ketocholesterol-containing nanoemulsion through LDL receptor reduces melanoma growth and : a preliminary report.

Oncotarget 2018 Mar 4;9(18):14160-14174. Epub 2018 Feb 4.

Laboratory of Genetics and Molecular Hematology (LIM31), Department of Hematology, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, Brazil.

Oxysterols are cholesterol oxygenated derivatives which possess several biological actions. Among oxysterols, 7-ketocholesterol (7KC) is known to induce cell death. Here, we hypothesized that 7KC cytotoxicity could be applied in cancer therapeutics. 7KC was incorporated into a lipid core nanoemulsion. As a cellular model the murine melanoma cell line B16F10 was used. The nanoparticle (7KCLDE) uptake into tumor cells was displaced by increasing amounts of low-density-lipoproteins (LDL) suggesting a LDL-receptor-mediated cell internalization. 7KCLDE was mainly cytostatic, which led to an accumulation of polyploid cells. Nevertheless, a single dose of 7KCLDE killed roughly 10% of melanoma cells. In addition, it was observed dissipation of the transmembrane potential, evidenced with flow cytometry; presence of autophagic vacuoles, visualized and quantified with flow cytometry and acridine orange; and presence of myelin figures, observed with ultrastructural microscopy. 7KCLDE impaired cytokenesis was accompanied by changes in cellular morphology into a fibroblastoid shape which is supported by cytoskeletal rearrangements, as shown by the increased actin polymerization. 7KCLDE was injected into B16 melanoma tumor-bearing mice. 7KCLDE accumulated in the liver and tumor. In melanoma tumor 7KCLDE promoted a >50% size reduction, enlarged the necrotic area, and reduced intratumoral vasculature. 7KCLDE increased the survival rates of animals, without hematologic or liver toxicity. Although more pre-clinical studies should be performed, our preliminary results suggested that 7KCLDE is a promising novel preparation for cancer chemotherapy.
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http://dx.doi.org/10.18632/oncotarget.24389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5865661PMC
March 2018

Quantitative evaluation of collagen and elastic fibers after intense pulsed light treatment of mouse skin.

Lasers Surg Med 2018 Jan 16. Epub 2018 Jan 16.

Laboratory of Biochemistry and Biophysics of Butantan Institute, University of Sao Paulo, Butantã, Sao Paulo, Brazil.

Background And Objective: The aging of human skin includes intrinsic aging and photo-aging, which are characterized by a decrease in collagen and the deposition of abnormal elastic fibers. Intense pulsed light (IPL) sources are widely used in medicine to treat various cosmetic problems, including photo-damaged skin. Few studies have examined the microscopic changes produced by IPL. The objective of this study was to quantitatively evaluate the effects of IPL on collagen and elastic fibers in mice.

Materials And Methods: Forty female BALB/c mice were divided into four subgroups. Group 1 was the control group (n = 10), and groups 2, 3, and 4 were treatment groups (n = 10 in each group). Group 2 received one treatment, group 3 received two treatments, and group 4 received three treatments every 2 weeks. Skin tissue was obtained from irradiated areas 24 hours after the last treatment in each mouse. Collagen fibers were identified using the picrosirius red method. Elastic fibers were marked by Weigert-oxone stain. All samples were analyzed and quantified by a light microscope using analyzer system images.

Results: Group 4, which received three IPL treatments, showed significant quantitative increases in both collagen fibers (P < 0.05) and elastic fibers (P < 0.01). Collagen fibers demonstrated a better parallel distribution in relation to the epidermis.

Conclusion: IPL treatment significantly increased the number of collagen and elastic fibers within the dermis and improved the parallel distribution of collagen fibers in relation to the epidermis. These results were evident after three IPL treatments. Lasers Surg. Med. © 2018 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/lsm.22782DOI Listing
January 2018

Egg and fourth instar larvae gut of Aedes aegypti as a source of stem cells.

Tissue Cell 2016 Oct 18;48(5):558-65. Epub 2016 May 18.

Departament of Surgery, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, SP, Brazil.

According to the World Health Organization, 2015 registered more than 1.206.172 cases of Dengue in the Americas. Recently, the Aedes aegypti has been not only related to Dengue, but also with cases of Zika virus and Chikungunya. Due to its epidemiological importance, this study characterized the morphology of the embryonated eggs of A. aegypti and provided a protocol to culture stem cells from eggs and digestive tract of fourth instar larvae in order to examine cell biology and expression of markers in these vectors. Cells were isolated and cultured in DMEM-High at 28°C, and their morphology, cell cycle and immunophenotyping were examined. Morphologically, embryos were at the end of the embryonic period and showed: head, thorax, and abdomen with eight abdominal segments. The embryonic tissues expressed markers related to cell proliferation (PCNA), pluripotency (Sox2 and OCT3/4), neural cells (Nestin), mesenchymal cells (Vimentin and Stro-1), and endosomal cells (GM130 and RAB5). In culture, cells from both tissues (eggs and larvae gut) were composed by a heterogeneous population. The cells had a globoid shape and small size. Cell cycle analysis on passage 1 (P1) showed 27.5%±2.0% of cell debris, 68% of cells on G0-G1 phase, 30.2% on S phase, 1.9%±0.5% on G2-M phase. In addition, cells on passage 2 showed: 10% of cell debris, 92.4% of cells on G0-G1 phase, 6.8% on S phase, 0.6% on G2-M phase. Embryonated eggs expressed markers involved with pluripotency (Sox2 and Oct 3/4), mesenchymal cells (vimentin and Stro-1), neural cells (Nestin), and cellular death by apoptosis (Caspase 3). Specific endosomal markers for insect cells (GM130 and RAB5) were also highly expressed. In cell culture of A. aegypti larvae gut the same labeling pattern was observed, with a small decrease in the expression of mesenchymal (vimentin and Stro-1) and neural (Nestin) markers. In summary, we were able to establish a protocol to culture embryonated eggs and larvae gut of A. aegypti, describing the characteristics of undifferentiated cells, as well as the cell cycle and expression of markers, which can be used for biotechnology studies for the biological control of this vector.
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http://dx.doi.org/10.1016/j.tice.2016.05.004DOI Listing
October 2016

Simvastatin increases the antineoplastic actions of paclitaxel carried in lipid nanoemulsions in melanoma-bearing mice.

Int J Nanomedicine 2016 7;11:885-904. Epub 2016 Mar 7.

Laboratory of Metabolism and Lipids, Heart Institute of the Medical School Hospital, University of São Paulo, São Paulo, Brazil; Department of Clinical Chemistry, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.

Purpose: Lipid nanoemulsions (LDEs) that bind to low-density lipoprotein (LDL) receptors used as carriers of paclitaxel (PTX) can decrease toxicity and increase PTX antitumoral action. The administration of simvastatin (Simva), which lowers LDL-cholesterol, was tested as an adjuvant to commercial PTX and to PTX associated with LDE (LDE-PTX).

Materials And Methods: B16F10 melanoma-bearing mice were treated with saline solution or LDE (controls), Simva, PTX, PTX and Simva, LDE-PTX, and LDE-PTX and Simva: PTX dose 17.5 μmol/kg (three intraperitoneal injections, 3 alternate days): Simva 50 mg/kg/day by gavage, 9 consecutive days.

Results: Compared with saline controls, 95% tumor-growth inhibition was achieved by LDE-PTX and Simva, 61% by LDE-PTX, 44% by PTX and Simva, and 43% by PTX. Simva alone had no effect. Metastasis developed in only 37% of the LDE-PTX and Simva, 60% in LDE-PTX, and 90% in PTX and Simva groups. Survival rates were higher in LDE-PTX and Simva and in LDE-PTX groups. The LDE-PTX and Simva group presented tumors with reduced cellular density and increased collagen fibers I and III. Tumors from all groups showed reduction in immunohistochemical expression of ICAM, MCP-1, and MMP-9; LDE-PTX and Simva presented the lowest MMP-9 expression. Expression of p21 was increased in the Simva, LDE-PTX, and LDE-PTX and Simva groups. In the Simva and LDE-PTX and Simva groups, expression of cyclin D1, a proliferation and survival promoter of tumor cells, was decreased. Therapy with LDE-PTX and Simva showed negligible toxicity compared with PTX and Simva, which resulted in weight loss and myelosuppression.

Conclusion: Simva increased the antitumor activity of PTX carried in LDE but not of PTX commercial presentation, possibly because statins increase the expression of LDL receptors that internalize LDE-PTX.
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http://dx.doi.org/10.2147/IJN.S88546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4788363PMC
October 2016

Comparative study of equine mesenchymal stem cells from healthy and injured synovial tissues: an in vitro assessment.

Stem Cell Res Ther 2016 Mar 5;7:35. Epub 2016 Mar 5.

Department of Internal Medicine, School of Veterinary Medicine and Animal Science, University of São Paulo (USP), Avenida Prof. Orlando Marques de Paiva, 87, 05508-270, São Paulo, SP, Brazil.

Background: Bone marrow and adipose tissues are known sources of mesenchymal stem cells (MSCs) in horses; however, synovial tissues might be a promising alternative. The aim of this study was to evaluate phenotypic characteristics and differentiation potential of equine MSCs from synovial fluid (SF) and synovial membrane (SM) of healthy joints (SF-H and SM-H), joints with osteoarthritis (SF-OA and SM-OA) and joints with osteochondritis dissecans (SF-OCD and SM-OCD) to determine the most suitable synovial source for an allogeneic therapy cell bank.

Methods: Expression of the markers CD90, CD105, CD44, and CD34 in SF-H, SM-H, SF-OA, SM-OA, SF-OCD and SM-OCD was verified by flow cytometry, and expression of cytokeratin, vimentin, PGP 9.5, PCNA, lysozyme, nanog, and Oct4 was verified by immunocytochemistry. MSCs were cultured and evaluated for their chondrogenic, osteogenic and adipogenic differentiation potential. Final quantification of extracellular matrix and mineralized matrix was determined using AxioVision software. A tumorigenicity test was conducted in Balb-C(nu/nu) mice to verify the safety of the MSCs from these sources.

Results: Cultured cells from SF and SM exhibited fibroblastoid morphology and the ability to adhere to plastic. The time elapsed between primary culture and the third passage was approximately 73 days for SF-H, 89 days for SF-OCD, 60 days for SF-OA, 68 days for SM-H, 57 days for SM-OCD and 54 days for SM-OA. The doubling time for SF-OCD was higher than that for other cells at the first passage (P < 0.05). MSCs from synovial tissues showed positive expression of the markers CD90, CD44, lysozyme, PGP 9.5, PCNA and vimentin and were able to differentiate into chondrogenic (21 days) and osteogenic (21 days) lineages, and, although poorly, into adipogenic lineages (14 days). The areas staining positive for extracellular matrix in the SF-H and SM-H groups were larger than those in the SF-OA and SM-OA groups (P < 0.05). The positive mineralized matrix area in the SF-H group was larger than those in all the other groups (P < 0.05). The studied cells exhibited no tumorigenic effects.

Conclusions: SF and SM are viable sources of equine MSCs. All sources studied provide suitable MSCs for an allogeneic therapy cell bank; nevertheless, MSCs from healthy joints may be preferable for cell banking purposes because they exhibit better chondrogenic differentiation capacity.
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http://dx.doi.org/10.1186/s13287-016-0294-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4779201PMC
March 2016

Evaluation of the Proliferative Effects Induced by Low-Level Laser Therapy in Bone Marrow Stem Cell Culture.

Photomed Laser Surg 2015 Dec 18;33(12):610-6. Epub 2015 Nov 18.

1 Postgraduate Department, Cruzeiro do Sul University São Paulo , SP, Brasil .

Objective: The objective of this study was to evaluate the effect of laser irradiation on dog bone marrow stem cells.

Background Data: Low doses of low-level red laser positively affect the viability of mesenchymal stem cells, and also increase proliferation.

Methods: Low-level laser (wavelength, 660 nm; power output, 50 mW), was applied to dog bone marrow stem cell cultures (DBMSC). The energy densities delivered varied from 1 to 12J/cm(2). The effect of the laser irradiation was evaluated on cell proliferation measured with the MTT colorimetric test, cell cycle phase, and on lipidic peroxidation (free radical production).

Results: The results indicate that laser irradiation to DBMSC did not change the morphology of the cells, but significantly increased their viability and the number of cells at the G2/M phase with 6, 10, and 12 J/cm(2). On the other hand, malonaldehyde production was significantly enhanced with 8 J/cm(2).

Conclusions: The parameters used to irradiate DBMSC increased significantly proliferation without producing high levels of reactive oxygen species (ROS).
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http://dx.doi.org/10.1089/pho.2014.3864DOI Listing
December 2015

Characterization of adipose-derived stem cells of anatomical region from mice.

BMC Res Notes 2014 Aug 20;7:552. Epub 2014 Aug 20.

Biochemistry and Biophysical Laboratory, Butantan Institute, 1500, Vital Brasil Avenue, Sao Paulo, Brazil.

Background: Stem cells constitute a group of great capacity for self-renewal, long-term viability, and multi-lineage potential. Several studies have provided evidence that adipose tissue represents an alternative source of stem cells, with the main benefit of adipose-derived stem cells being that they can be easily harvested from patients by a simple minimally invasive method and can be easily cultured. The aim of this study was to establish a culture protocol for obtaining and characterizing adipose-derived stem cells (ADSCs) from C57BL/6 J mice.

Results: The results showed that the yield, viability, and cell morphology obtained differ according to the age of isolated anatomic regions of the adipose tissue from ovarian and epididymis. The results of determination of cyclin D1 showed uniformity in the expression between different populations of ADSCs. A significant increase in the expression of caspase-3 active, was also observed in large cell populations from mice after 120 days. ADSCs were positive for mesenchymal markers CD90 and CD105, Nanog, SSEA-1, CD106, and VEGFR-1, and negative for hematopoietic markers CD34 and CD45. A large number of cells in S + G2/M phases was also observed for both sexes, demonstrating high proliferative capacity of ADSCs.

Conclusions: We observed that the adipose tissue of C57BL/6 J mice, isolated from the studied anatomic regions, is a promising source for obtaining pluripotent mesenchymal stem cells with high viability and proliferative response.
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http://dx.doi.org/10.1186/1756-0500-7-552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4156637PMC
August 2014

Association of daunorubicin to a lipid nanoemulsion that binds to low-density lipoprotein receptors enhances the antitumour action and decreases the toxicity of the drug in melanoma-bearing mice.

J Pharm Pharmacol 2014 Dec 17;66(12):1698-709. Epub 2014 Aug 17.

Lipid Metabolism Laboratory, Heart Institute of the Medical School Hospital, University of São Paulo, São Paulo, Brazil.

Objectives: To test the toxicity and antitumoral activity of the compound N-oleyl-daunorubicin (oDNR) with a cholesterol-rich nanoemulsion (LDE) formulation.

Methods: LDE-oDNR was prepared by high-pressure homogenisation of lipid mixtures. B16F10 melanoma cells and NIH/3T3 fibroblasts were used for cytotoxicity tests. The maximum tolerated dose (MTD) of both commercial and LDE-oDNR was determined in mice, and melanoma-bearing mice were used for the antitumoral activity tests.

Key Findings: CC50 for LDE-oDNR and DNR in melanoma cells were 200 μm and 15 μm, respectively, but LDE-oDNR was less toxic against fibroblasts than DNR. MTD for LDE-oDNR was 65-fold higher than commercial DNR. In tumour-bearing mice, LDE-oDNR (7.5 μmol/kg) reduced tumour growth by 59 ± 2%, whereas the reduction by DNR was only 23 ± 2%. LDE-oDNR increased survival rates (P < 0.05), which was not achieved by DNR treatment. The number of mice with metastasis was only 30% in LDE-oDNR-treated mice, compared with 82% under DNR treatment. By flow cytometry, there were 9% viable cells in tumours of animals treated with LDE-oDNR compared with 27% in DNR-treated animals. Less haematological toxicity was observed in LDE-oDNR-treated mice.

Conclusions: Compared with DNR, LDE-oDNR improved tumour growth inhibition and survival rates with pronouncedly less toxicity, and thus may become a new tool for cancer treatment.
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http://dx.doi.org/10.1111/jphp.12296DOI Listing
December 2014

Angiogenesis enhanced by Phyllocaulis boraceiensis mucus in human cells.

FEBS J 2013 Oct 12;280(20):5118-27. Epub 2013 Sep 12.

Laboratory of Biochemistry and Biophysics, Butantan Institute, São Paulo, Brazil.

Phyllocaulis boraceiensis mucus is known to be a compound capable of inducing cell proliferation and enhancing the wound healing process. The process of angiogenesis is a chain of mechanisms responsible for the formation of new vessels, which are are involved in cell proliferation, and factors that will act in the healing process. Our aim was to demonstrate that the angiogenesis process is enhanced in cultures of endothelial cells and fibroblasts treated with P. boraceiensis mucus. Experiments were carried out with 10(5) cells·mL(-1) of endothelial cells and fibroblasts treated with P. boraceiensis mucus in concentrations that have significant effects in proliferation assays, i.e. 0.012 μg·μL(-1) and 0.18 μg·μL(-1) , both of which cause extreme responses. Aliquots of 100 μL of cell suspensions were incubated for 1 h at 4 °C with 1 μL of antibodies specific for the cell markers vascular endothelial growth factor receptor 1 and cluster of differentiation 34, and negative isotype controls. Reading and expression analysis of cell markers was performed on a FACSCalibur flow cytometer. Expression levels of vascular endothelial growth factor receptor 1 and cluster of differentiation 34 expression were significantly increased in endothelial cells cultivated with 0.012 μg·μL(-1) P. boraceiensis mucus, suggesting that this compound is capable of enhancing angiogenesis.
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http://dx.doi.org/10.1111/febs.12487DOI Listing
October 2013

Drug-targeting in combined cancer chemotherapy: tumor growth inhibition in mice by association of paclitaxel and etoposide with a cholesterol-rich nanoemulsion.

Cell Oncol (Dordr) 2012 Dec 3;35(6):451-60. Epub 2012 Oct 3.

Lipid Metabolism Laboratory, the Heart Institute (INCOR) of the Medical School Hospital, University of São Paulo, São Paulo, Brazil.

Background: Lipid nanoemulsions (LDE) may be used as carriers of paclitaxel (PTX) and etoposide (ETP) to decrease toxicity and increase the therapeutic action of those drugs. The current study investigates the combined chemotherapy with PTX and ETP associated with LDE.

Methods: Four groups of 10-20 B16F10 melanoma-bearing mice were treated with LDE-PTX and LDE-ETP in combination (LDE-PTX + ETP), commercial PTX and ETP in combination (PTX + ETP), single LDE-PTX, and single LDE-ETP. PTX and ETX doses were 9 μmol/kg administered in three intraperitoneal injections on three alternate days. In two control groups mice were treated with saline solution or LDE alone. Tumor growth, metastasis presence, cell-cycle distribution, blood cell counts and histological data were analyzed. Toxicity of all treatments was evaluated in mice without tumors.

Results: Tumor growth inhibition was similarly strong in all treatment groups. However, there was a greater reduction in the number of animals bearing metastases in the LDE-PTX + ETP group (30 %) in comparison to the PTX + ETP group (82 %, p < 0.05). Reduction of cellular density, blood vessels and increase of collagen fibers in tumor tissues were observed in the LDE-PTX + ETP group but not in the PTX + ETP group, and in both groups reduced melanoma-related anemia and thrombocytosis were observed. Flow cytometric analysis suggested that LDE-PTX + ETP exhibited greater selectivity to neoplastic cells than PTX-ETP, showing arrest (65 %) in the G(2)/M phase of the cell cycle (p < 0.001). Toxicity manifested by weight loss and myelosuppression was markedly milder in the LDE-PTX + ETP than in the PTX + ETP group.

Conclusion: LDE-PTX + ETP combined drug-targeting therapy showed markedly superior anti-cancer properties and reduced toxicity compared to PTX + ETP.
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http://dx.doi.org/10.1007/s13402-012-0104-6DOI Listing
December 2012

Azidothymidine is effective against human multiple myeloma: a new use for an old drug?

Anticancer Agents Med Chem 2013 Jan;13(1):186-92

Laboratory of Genetics and Molecular Hematology, LIM 31, University of São Paulo School of Medicine, São Paulo, Brazil.

Azidothymidine (AZT) is an antiretroviral drug that affects cell proliferation, apoptosis, and the NF-κB pathway. As multiple myeloma (MM) presents with constitutive activation of NF-κB, we analyzed the effect of AZT on human MM cell lines. We evaluated the cytotoxic effect of AZT in human MM cell lines sensitive (8226/S) or resistant to doxorubicin (8226/DX5) and human T cell lymphoblast-like cells, uterine sarcoma cells, and HUVEC using MTT assay. Cytotoxicity was also evaluated in vivo in nude mice xenografted with 8226/S tumor. The effect of AZT on the expression of genes involved in cell proliferation, apoptosis, angiogenesis, and the NF-κB pathway was analyzed in the xenografts using real-time polymerase chain reaction. AZT was effective against both 8226/S and 8226/DX5 cells in a dose and time-dependent manner (p = 0.02) in vitro and promoted cell cycle arrest in S phase in these cells. The tumor volume was lower in mice treated with AZT compared to untreated mice (p = 0.0003). AZT down-regulated the pro-proliferative genes encoding AKT1, MYC, STAT1, MAPK8, MAPK9, CCL-3, Bcl-3, and cyclin D2; pro-angiogenenic genes encoding VEGF and IL8; and genes involved in cell adhesion (ICAM1 and FN1) and the NF-κB pathway. AZT up-regulated the expression of tumor suppressor gene FOXP1 and the pro-apoptotic genes encoding BID, Bcl-10, and caspase-8. Thus, we demonstrated the cytotoxic effect of AZT in human MM cell lines for the first time. Our data may provide the rationale for future clinical trials of AZT for treating MM.
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January 2013

A lipocalin-derived Peptide modulating fibroblasts and extracellular matrix proteins.

J Toxicol 2012 26;2012:325250. Epub 2012 Apr 26.

Laboratory of Biochemistry and Biophysics, Butantan Institute, Avenida Vital Brasil 1500, 05503-900 São Paulo, SP, Brazil.

Lipocalin family members have been implicated in development, regeneration, and pathological processes, but their roles are unclear. Interestingly, these proteins are found abundant in the venom of the Lonomia obliqua caterpillar. Lipocalins are β-barrel proteins, which have three conserved motifs in their amino acid sequence. One of these motifs was shown to be a sequence signature involved in cell modulation. The aim of this study is to investigate the effects of a synthetic peptide comprising the lipocalin sequence motif in fibroblasts. This peptide suppressed caspase 3 activity and upregulated Bcl-2 and Ki-67, but did not interfere with GPCR calcium mobilization. Fibroblast responses also involved increased expression of proinflammatory mediators. Increase of extracellular matrix proteins, such as collagen, fibronectin, and tenascin, was observed. Increase in collagen content was also observed in vivo. Results indicate that modulation effects displayed by lipocalins through this sequence motif involve cell survival, extracellular matrix remodeling, and cytokine signaling. Such effects can be related to the lipocalin roles in disease, development, and tissue repair.
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http://dx.doi.org/10.1155/2012/325250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3379166PMC
August 2012

Novel formulation of a methotrexate derivative with a lipid nanoemulsion.

Int J Nanomedicine 2011 12;6:2285-95. Epub 2011 Oct 12.

Heart Institute of the Medical School Hospital, University of São Paulo, São Paulo, Brazil.

Background: Lipid nanoemulsions that bind to low-density lipoprotein receptors can concentrate chemotherapeutic agents in tissues with low-density lipoprotein receptor overexpression and decrease the toxicity of the treatment. The aim of this study was to develop a new formulation using a lipophilic derivative of methotrexate, ie, didodecyl methotrexate (ddMTX), associated with a lipid nanoemulsion (ddMTX-LDE).

Methods: ddMTX was synthesized by an esterification reaction between methotrexate and dodecyl bromide. The lipid nanoemulsion was prepared by four hours of ultrasonication of a mixture of phosphatidylcholine, triolein, and cholesteryloleate. Association of ddMTX with the lipid nanoemulsion was performed by additional cosonication of ddMTX with the previously prepared lipid nanoemulsion. Formulation stability was evaluated, and cell uptake, cytotoxicity, and acute animal toxicity studies were performed.

Results: The yield of ddMTX incorporation was 98% and the particle size of LDE-ddMTX was 60 nm. After 48 hours of incubation with plasma, approximately 28% ddMTX was released from the lipid nanoemulsion. The formulation remained stable for at least 45 days at 4°C. Cytotoxicity of LDE-ddMTX against K562 and HL60 neoplastic cells was higher than for methotrexate (50% inhibitory concentration [IC(50)] 1.6 versus 18.2 mM and 0.2 versus 26 mM, respectively), and cellular uptake of LDE-ddMTX was 90-fold higher than that of methotrexate in K562 cells and 75-fold in HL60 cells. Toxicity of LDE-ddMTX, administered at escalating doses, was higher than for methotrexate (LD(50) 115 mg/kg versus 470 mg/kg; maximum tolerated dose 47 mg/kg versus 94 mg/kg) in mice. However, the hematological toxicity of LDE-ddMTX was lower than for methotrexate.

Conclusion: LDE-ddMTX was stable, and uptake of the formulation by neoplastic cells was remarkably greater than of methotrexate, which resulted in markedly greater cytotoxicity. LDE-ddMTX is thus a promising formulation to be tested in future animal models of cancer or rheumatic disease, wherein methotrexate is widely used.
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http://dx.doi.org/10.2147/IJN.S18039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3205125PMC
May 2012

Antitumor potential induction and free radicals production in melanoma cells by Boron Neutron Capture Therapy.

Appl Radiat Isot 2011 Dec 20;69(12):1748-51. Epub 2011 May 20.

Biochemical and Biophysical Laboratory, Butantan Institute, São Paulo, Brazil.

Antiproliferative and oxidative damage effects occurring in Boron Neutron Capture Therapy (BNCT) in normal fibroblasts and melanoma cell lines were analyzed. Melanoma cells and normal fibroblasts were treated with different concentrations of Boronophenylalanine and irradiated with thermal neutron flux. The cellular viability and the oxidative stress were determined. BNCT induced free radicals production and proliferative potential inhibition in melanoma cells. Therefore, this therapeutic technique could be considered efficient to inhibit growth of melanoma with minimal effects on normal tissues.
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http://dx.doi.org/10.1016/j.apradiso.2011.05.017DOI Listing
December 2011

Boron neutron capture therapy induces cell cycle arrest and DNA fragmentation in murine melanoma cells.

Appl Radiat Isot 2011 Dec 21;69(12):1741-4. Epub 2011 Mar 21.

Biochemical and Biophysical Laboratory, Butantan Institute, São Paulo, Brazil.

The melanoma is a highly lethal skin tumor, with a high incidence. Boron Neutron Capture Therapy (BNCT) is a radiotherapy which combines Boron with thermal neutrons, constituting a binary system. B16F10 melanoma and L929 fibroblasts were treated with Boronophenylalanine and irradiated with thermal neutron flux. The electric potential of mitochondrial membrane, cyclin D1 and caspase-3 markers were analyzed. BNCT induced a cell death increase and cyclin D1 amount decreased only in B16F10 melanoma. Besides, there was not caspase-3 phosphorylation.
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http://dx.doi.org/10.1016/j.apradiso.2011.03.005DOI Listing
December 2011

Simvastatin impairs murine melanoma growth.

Lipids Health Dis 2010 Dec 16;9:142. Epub 2010 Dec 16.

State University of Ponta Grossa, Biological and Health Science Multidisciplinary Laboratory, Ponta Grossa, Brazil.

Background: Statins induces cell cycle arrest, apoptosis, reduction of angiogenic factors, inhibition of the endothelial growth factor, impairing tissue adhesion and attenuation of the resistance mechanisms. The aim of this study was evaluate the anti-tumoral activity of simvastatin in a B16F10 melanoma-mouse model.

Methods: Melanoma cells were treated with different concentrations of simvastatin and assessed by viability methods. Melanoma cells (5 × 10(4)) were implanted in two month old C57Bl6/J mice. Around 7 days after cells injection, the oral treatments were started with simvastatin (5 mg/kg/day, p.o.). Tumor size, hematological and biochemical analyses were evaluated.

Results: Simvastatin at a concentration of 0.8 μM, 1.2 μM and 1.6 μM had toxic effect. Concentration of 1.6 μM induced a massive death in the first 24 h of incubation. Simvastatin at 0.8 μM induces early cell cycle arrest in G0/G1, followed by increase of hypodiploidy. Tumor size were evaluated and the difference of treated group and control, after ten days, demonstrates that simvastatin inhibited the tumor expansion in 68%.

Conclusion: Simvastatin at 1.6 μM, presented cytototoxicity after 72 h of treatment, with an intense death. In vivo, simvastatin being potentially useful as an antiproliferative drug, with an impairment of growth after ten days.
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http://dx.doi.org/10.1186/1476-511X-9-142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3012033PMC
December 2010

Modification of composition of a nanoemulsion with different cholesteryl ester molecular species: effects on stability, peroxidation, and cell uptake.

Int J Nanomedicine 2010 Sep 20;5:679-86. Epub 2010 Sep 20.

Lipid Metabolism Laboratory, Heart Institute (InCor), Medical School Hospital, University of São Paulo, São Paulo, Brazil.

Purpose: Use of lipid nanoemulsions as carriers of drugs for therapeutic or diagnostic purposes has been increasingly studied. Here, it was tested whether modifications of core particle constitution could affect the characteristics and biologic properties of lipid nanoemulsions.

Methods: Three nanoemulsions were prepared using cholesteryl oleate, cholesteryl stearate, or cholesteryl linoleate as main core constituents. Particle size, stability, pH, peroxidation of the nanoemulsions, and cell survival and uptake by different cell lines were evaluated.

Results: It was shown that cholesteryl stearate nanoemulsions had the greatest particle size and all three nanoemulsions were stable during the 237-day observation period. The pH of the three nanoemulsion preparations tended to decrease over time, but the decrease in pH of cholesteryl stearate was smaller than that of cholesteryl oleate and cholesteryl linoleate. Lipoperoxidation was greater in cholesteryl linoleate than in cholesteryl oleate and cholesteryl stearate. After four hours' incubation of human umbilical vein endothelial cells (HUVEC) with nanoemulsions, peroxidation was minimal in the presence of cholesteryl oleate and more pronounced with cholesteryl linoleate and cholesteryl stearate. In contrast, macrophage incubates showed the highest peroxidation rates with cholesteryl oleate. Cholesteryl linoleate induced the highest cell peroxidation rates, except in macrophages. Uptake of cholesteryl oleate nanoemulsion by HUVEC and fibroblasts was greater than that of cholesteryl linoleate and cholesteryl stearate. Uptake of the three nanoemulsions by monocytes was equal. Uptake of cholesteryl oleate and cholesteryl linoleate by macrophages was negligible, but macrophage uptake of cholesteryl stearate was higher. In H292 tumor cells, cholesteryl oleate showed the highest uptakes. HUVEC showed higher survival rates when incubated with cholesteryl stearate and smaller survival with cholesteryl linoleate. H292 survival was greater with cholesteryl stearate.

Conclusion: Although all three nanoemulsion types were stable for a long period, considerable differences were observed in size, oxidation status, and cell survival and nanoemulsion uptake in all tested cell lines. Those differences may be helpful in protocol planning and interpretation of data from experiments with lipid nanoemulsions.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2948947PMC
http://dx.doi.org/10.2147/ijn.s12293DOI Listing
September 2010

Synthetic nanoemulsion resembling a protein-free model of 7-ketocholesterol containing low density lipoprotein: In vitro and in vivo studies.

Biol Res 2010 1;43(4):439-44. Epub 2011 Feb 1.

Laboratory of Lipid Metabolism, Instituto do Coração, Hospital das Clinicas, Faculdade de Medicina, Universidade de São Paulo, Av. Dr. Enéas de Carvalho Aguiar 155, São Paulo, SP, Brazil.

7-ketocholesterol (7-KC) differs from cholesterol by a functional ketone group at C7. It is an oxygenated cholesterol derivative (oxysterol), commonly present in oxidized low-density lipoprotein (LDL). Oxysterols are generated and participate in several physiologic and pathophysiologic processes. For instance, the cytotoxic effects of oxidized LDL have been widely attributed to bioactive compounds like oxysterols. The toxicity is in part due to 7-KC. Here we aimed to demonstrate the possibility of incorporating 7-KC into the synthetic nanoemulsion LDE, which resembles LDL in composition and behavior. This would provide a suitable artificial particle resembling LDL to study 7-KC metabolism. We were able to incorporate 7-KC in several amounts into LDE. The incorporation was evaluated and confirmed by several methods, including gel filtration chromatography, using radiolabeled lipids. The incorporation did not change the main lipid composition characteristics of the new nanoparticle. Particle sizes were also evaluated and did not differ from LDE. In vivo studies were performed by injecting the nanoemulsion into mice. The plasma kinetics and the targeted organs were the same as described for LDE. Therefore, 7-KC-LDE maintains composition, size and some functional characteristics of LDE and could be used in experiments dealing with 7-ketocholesterol metabolism in lipoproteins.
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http://dx.doi.org//S0716-97602010000400008DOI Listing
July 2012

Static electric fields interfere in the viability of cells exposed to ionising radiation.

Int J Radiat Biol 2009 Apr;85(4):314-21

Physics Institute, University of São Paulo, São Paulo, SP, Brazil.

Purpose: The interference of electric fields (EF) with biological processes is an issue of considerable interest. No studies have as yet been reported on the combined effect of EF plus ionising radiation. Here we report studies on this combined effect using the prokaryote Microcystis panniformis, the eukaryote Candida albicans and human cells.

Materials And Methods: Cultures of Microcystis panniformis (Cyanobacteria) in glass tubes were irradiated with doses in the interval 0.5-5 kGy, using a (60)Co gamma source facility. Samples irradiated with 3 kGy were exposed for 2 h to a 20 V . cm(-1) static electric field and viable cells were enumerated. Cultures of Candida albicans were incubated at 36 degrees C for 20 h, gamma-irradiated with doses from 1-4 kGy, and submitted to an electric field of 180 V . cm(-1). Samples were examined under a fluorescence microscope and the number of unviable (red) and viable (apple green fluorescence) cells was determined. For crossing-check purposes, MRC5 strain of lung cells were irradiated with 2 Gy, exposed to an electric field of 1250 V/cm, incubated overnight with the anti-body anti-phospho-histone H2AX and examined under a fluorescence microscope to quantify nuclei with gamma-H2AX foci.

Results: In cells exposed to EF, death increased substantially compared to irradiation alone. In C. albicans we observed suppression of the DNA repair shoulder. The effect of EF in growth of M. panniformis was substantial; the number of surviving cells on day-2 after irradiation was 12 times greater than when an EF was applied. By the action of a static electric field on the irradiated MRC5 cells the number of nuclei with gamma-H2AX foci increased 40%, approximately.

Conclusions: Application of an EF following irradiation greatly increases cell death. The observation that the DNA repair shoulder in the survival curve of C. albicans is suppressed when cells are exposed to irradiation + EF suggests that EF likely inactivate cellular recovering processes. The result for the number of nuclei with gamma-H2AX foci in MRC5 cells indicates that an EF interferes mostly in the DNA repair mechanisms. A molecular ad-hoc model is proposed.
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http://dx.doi.org/10.1080/09553000902781121DOI Listing
April 2009

Patterns of cell proliferation and apoptosis by topographic region in normal Bos taurus vs. Bos indicus crossbreeds bovine placentae during pregnancy.

Reprod Biol Endocrinol 2009 Mar 30;7:25. Epub 2009 Mar 30.

Department of Surgery of the São Paulo University, Faculty of Veterinary Medicine and Animal Science, São Paulo, SP, 05508-270, Brazil.

Background: Placental and fetal growth requires high rates of cellular turnover and differentiation, which contributes to conceptus development. The trophoblast has unique properties and a wide range of metabolic, endocrine and angiogenic functions, but the proliferative profile of the bovine placenta characterized by flow cytometry analysis and its role in fetal development are currently uncharacterized. Complete understanding of placental apoptotic and proliferative rates may be relevant to development, especially if related to the pathogenesis of pregnancy losses and placental abnormalities.

Methods: In this study, the proliferation activity and apoptosis in different regions of normal bovine placenta (central and boundary regions of placentomes, placentomal fusion, microplacentomes, and interplacentomal regions), from distinct gestation periods (Days 70 to 290 of pregnancy), were analyzed by flow cytometry.

Results: Our results indicated that microplacentomes presented a lower number of apoptotic cells throughout pregnancy, with a higher proliferative activity by the end of gestation, suggesting that such structures do not contribute significantly to normal of placental functions and conceptus development during pregnancy. The placentome edges revealed a higher number of apoptotic cells from Day 170 on, which suggests that placentome detachment may well initiate in this region.

Conclusion: Variations involving proliferation and apoptotic rates may influence placental maturation and detachment, compromising placental functions and leading to fetal stress, abnormalities in development and abortion, as frequently seen in bovine pregnancies from in vitro fertilization and cloning procedures. Our findings describing the pattern of cell proliferation and apoptosis in normal bovine pregnancies may be useful for unraveling some of the developmental deviations seen in nature and after in vitro embryo manipulations.
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http://dx.doi.org/10.1186/1477-7827-7-25DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2669090PMC
March 2009

Analysis of cell cycle phases and proliferative capacity in mice bearing melanoma maintained on different dietary proteins.

J Cutan Pathol 2009 Oct 27;36(10):1053-62. Epub 2009 Jan 27.

Department of Food and Nutrition, State University of Campinas (UNICAMP), Campinas, Brazil.

Background: Diet seems to represent, directly or indirectly, 35% of all cancer reports. In this study, the influence of dietary protein on the growth of melanoma B16F10 was evaluated through analyses of cell cycle phases and proliferative capacity.

Methods: Flow cytometry and argyrophilic nucleolar organizer regions (AgNORs) technique were applied in mice bearing B16F10 melanoma cells fed on different dietary proteins. All data were submitted to statistical analyses.

Results: The G0/G1 phase increased for the animal groups fed bovine collagen hydrolysate (BCH) or BCH-P1 + whey protein isolate (WPI), compared with mice receiving only WPI, for all dietary groups treated and nontreated with paclitaxel. Mice that received BCH + WPI treated with paclitaxel showed the highest percentage of apoptosis compared with WPI group. AgNORs, total nucleolar organizer regions (NORs)/cells and dot number/cell for all dietary protein groups nontreated with paclitaxel were higher than for the WPI. The only two dietary protein groups treated with paclitaxel that presented higher total NORs and dot number/cell than the WPI group were BCH + WPI and BCH-P1 + WPI.

Conclusions: A significantly lower proliferative capacity and larger number of cells in the G0/G1 phase were observed for the dietary protein groups combining the two collagen hydrolysates, BCH or BCH-P1 with WPI, treated with paclitaxel.
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http://dx.doi.org/10.1111/j.1600-0560.2008.01220.xDOI Listing
October 2009

Toxin jararhagin in low doses induces interstitial edema and increases the metabolic rate and red blood cells in mice.

Toxicon 2006 Dec 14;48(8):1060-7. Epub 2006 Sep 14.

Departamento de Zoologia, Instituto de Biociências, Universidade Estadual Paulista (UNESP), Av. 24-A 1515, CEP 13506-900, Rio Claro, SP, Brazil.

Jararhagin is a metalloproteinase from Bothrops jararaca responsible for hemorrhage, inflammation, necrosis and edema. Effects of low doses of the toxin were analyzed on the energy metabolism of mice as well as its physiological implications. Measures of O(2) consumption (VO(2)) were quantified after 4 and 24h of the jararhagin administration during four weeks. Hematocrit and histology of the lungs were also analyzed after the end of the treatment. Results showed that animals that received subcutaneous doses of jararhagin had significant increase in VO(2) from second (120 ng) and third weeks (60 ng) after 4 and 24h, comparing to control, as well as in the number of erythrocytes after four weeks. Histology of the lungs showed interstitial edema within the alveolar septum. Results suggest that the jararhagin toxin caused an increase in VO(2) and edema of intra-alveolar septum. The increase of the erythrocytes could be a physiological response to adjust the higher necessity of oxygen, due to diffusional abnormalities caused by the edema. Thus, low doses of jararhagin promote endothelial edema which lead to changes in several physiological conditions.
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http://dx.doi.org/10.1016/j.toxicon.2006.08.014DOI Listing
December 2006

Evaluation in melanoma-bearing mice of an etoposide derivative associated to a cholesterol-rich nano-emulsion.

J Pharm Pharmacol 2006 Jun;58(6):801-8

Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.

A cholesterol-rich nano-emulsion (LDE) may be used as a vehicle to target antineoplastic drugs against cancer cells. The association of an etoposide derivative to LDE is stable and retains the cytotoxic activity of etoposide. We have evaluated the toxicity and antitumoral action of this new preparation in-vivo. Melanoma-bearing mice and control mice were administered LDE-etoposide oleate or commercial etoposide, either with or without radioactive labelling. The maximum tolerated dose (MTD), tissue distribution, plasma decay curves, pharmacokinetic parameters and antitumoral activity were determined. Association to LDE drastically reduced the drug toxicity, since MTD was approximately five-fold greater than in commercial etoposide. LDE-etoposide oleate was concentrated four-fold in the tumour compared with the normal adjacent tissues, was removed faster from plasma in tumour-bearing mice than in controls, and remained in the bloodstream longer than commercial etoposide. The tumour growth inhibition rate and survival were greater in animals treated with LDE-etoposide oleate compared with commercial etoposide. However, increasing the dose from 17 to 85 microM kg(-1) did not result in further improvement of the antitumour action. The incorporation of etoposide oleate to LDE resulted in markedly reduced toxicity and superior antitumoral activity. LDE-etoposide oleate is a promising new weapon for cancer treatment.
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http://dx.doi.org/10.1211/jpp.58.6.0010DOI Listing
June 2006

Nucleolar organizer region staining patterns in paraffin-embedded tissue cells from human skin cancers.

J Cutan Pathol 2005 May;32(5):323-8

Genetics Laboratory, Butantan Institute, São Paulo, SP, Brazil.

Background: Increased number of nucleoli (nucleolar organizer regions, NORs) with abnormal shapes and sizes, including small dots, has been used as prognostic tools to evaluate tumor proliferation levels and troublesome borderline lesions. In this study, NOR patterns of skin cancers were performed in the search of a valuable prognostic method to complement other histological procedures.

Methods: Paraffin-embedded tumor tissue was obtained from basal and squamous cell carcinomas, cutaneous malignant melanoma, premalignant lesions, and Skmel-28 human melanoma cells. Slices were dewaxed and AgNOR stained. The patterns were scored and submitted for statistical analyses.

Results: All types of cancer cells showed variable numbers of abnormally shaped nucleoli and dot-like structures. Only tumor cells presented four or more nucleoli, with or without dots, while 85% of the normal cells had one single NOR without dots. Most data were statistically significant when compared to normal cells. As a whole, squamous cell carcinoma and malignant melanoma tumor cells had less NOR alterations than basal cell carcinoma (BCC) tumor types.

Conclusions: Changes in the number and shape of nucleoli present in malignant cells could be attributed to increased levels on rDNA transcription on cancer cells, besides abnormal remodeling of chromatin, which could disrupt proper nucleoli association. Increased genetic alterations on malignant basal cells could contribute to impair invasive and migration abilities of BCC tumors.
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http://dx.doi.org/10.1111/j.0303-6987.2005.00322.xDOI Listing
May 2005

Improvement of paclitaxel therapeutic index by derivatization and association to a cholesterol-rich microemulsion: in vitro and in vivo studies.

Cancer Chemother Pharmacol 2005 Jun 22;55(6):565-76. Epub 2005 Feb 22.

Lipid Metabolism Laboratory, Heart Institute (InCor), University of São Paulo Medical School Hospital, Av. Dr. Eneas de Carvalho Aguiar, 44, 1 subsolo, 05403-000 Sao Paulo, Brazil.

A cholesterol-rich microemulsion or nanoparticle termed LDE concentrates in cancer tissues after injection into the bloodstream. Here the cytotoxicity, pharmacokinetics, toxicity to animals and therapeutic action of a paclitaxel lipophilic derivative associated to LDE is compared with those of the commercial paclitaxel. Results show that LDE-paclitaxel oleate is stable. The cytostatic activity of the drug in the complex is diminished compared with the commercial paclitaxel due to the cytotoxicity of the vehicle Cremophor EL used in the commercial formulation. Competition experiments in neoplastic cultured cells show that paclitaxel oleate and LDE are internalized together by the LDL receptor pathway. LDE-paclitaxel oleate arrests the G(2)/M phase of cell cycle, similarly to commercial paclitaxel. Tolerability to mice is remarkable, such that the lethal dose (LD(50)) was ninefold greater than that of the commercial formulation (LD(50) = 326 microM and 37 microM, respectively). LDE concentrates paclitaxel oleate in the tumor roughly fourfold relative to the normal adjacent tissues. At equimolar doses, the association of paclitaxel oleate with LDE results in remarkable changes in the drug pharmacokinetic parameters when compared to commercial paclitaxel (t(1/2)=218 min and 184 min, AUC=1,334 microg h/ml and 707 microg h/ml and CL=0.125 ml/min and 0.236 ml/min, respectively). Finally, the therapeutic efficacy of the complex is pronouncedly greater than that of the commercial paclitaxel, as indicated by the reduction in tumor growth, increase in survival rates and % cure of treated mice. In conclusion, LDE-paclitaxel oleate is a stable complex and compared with paclitaxel toxicity is considerably reduced and activity is enhanced, which may lead to improved therapeutic index in clinical use.
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http://dx.doi.org/10.1007/s00280-004-0930-yDOI Listing
June 2005